Eukaryotic chromatin is organized into loop domains, which might have both structural and functional roles such as in developmental stage- and cell type-specific gene expression and replication. It is believed that chromatin loops are anchored to the nuclear substructure (or matrix) at specific DNA regions called matrix attachment regions (MARs). SATB1 specifically binds in vivo to specific DNA sites with an ATC sequence context, which has high propensity to unwind under negative superhelical strain. These sites are referred to as base-unpairing region (BUR) and are hyper-reactive to chemical carcinogens. BURs represent a hallmark of MARs. SATB1 is a cell-type specific BUR-binding protein predominantly expressed in thymocytes. Analyses of SATB1 knockout mice showed that SATB1 is essential for proper T cell development by orchestrating spatial and temporal expression of a large number of genes in thymocytes. SATB1 actively tethers SATB1-bound sequences in vivo onto the nuclear substructure and regulates distant genes. Our recent data using SATB1 knockout mice and ES cells show that SATB1 has a regulatory role in imprinted genes, Igf2 and H19, as well as Xist, In the current proposal, we propose to 1) study the role of SATB1 in regulating imprinted Igf2 and H19 genes, 2) determine the effects of deleting an in vivo SATB1-binding sequence BUR located between Igf2 and H19 on the allele-specific expression, methylation and chromatin structure of Igf2 and H19 genes in mice, 3) examine the role of SATBI in the coordinated expression of Xist and its antisense gene Tsix, 4) determine whether BURs have a role in the chromosomal replicator function, and 5) isolate a BUR-binding protein distinct from SATB1 from ES cells possessing the activity similar to SATB1 in assembling the chromatin-remodeling complex. These experiments, using both SATB1 knockout mice and ES cells will provide important information on not only the biological function of SATB1, but also how higher order chromatin structure is organized to regulate gene expression and replication through DNA loop anchorage sequences.